Johne's disease, paratuberculosis (Ptb), is a chronic wasting disease of the intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). It causes significant economic loss to animal producers, especially in the dairy industry, due to increase in forage consumption, decreased milk production, and early culling due to poor health of affected animals (6, 22, 28). The disease has been difficult to control because of the lack of sensitive specific diagnostic assays and the lack of an efficacious vaccine. Available assays such as Map-antigen ELISAs and the IFN-γ assays vary in their capacity to detect infected animals in the early stages of the disease (36). Available vaccines have been shown to reduce the severity of pathology but not stop shedding of bacteria (18). Consequently, there is a continuing need to develop better diagnostic assays and also a better vaccine that, at a minimum, stops shedding during the productive life of dairy cattle.
An important prerequisite to control this disease is understanding the molecular mechanisms of Map pathogensis. To increase our knowledge of the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines, an efficient method for generating targeted gene knockouts is urgently needed. In contrast to the successful gene disruption in fast-growing mycobacteria such as M. smegmatis (8, 10, 24, 33), gene disruption in slow-growing mycobacteria has traditionally proven inefficient, partly due to high frequency of illegitimate recombination and their characteristic aggregation in culture that makes isolation of individual clones problematic (1, 23, 26).
Recent major advances in the methods of genetic manipulation have overcome some of the difficulties encountered in attempting to disrupt genes in slow-growing mycobacteria. The ability to selectively disrupt genes of interest has improved our understanding of pathogenic mycobacterial virulence based on specific gene function. For example, allelic exchange using either linear DNA fragments or suicide vectors, insertion mutagenesis using transposons, and specialized transduction have been successful in M. tuberculosis and M. bovis (2-4, 7, 11). Although random transposon mutagenesis has been reported in Map (12, 19, 32), directed allelic exchange mutagenesis has still remained intractable. This inability to inactivate specific genes has impeded progress in the use of the recently completed genome sequence of Map K10 (25). A new methodology to generate allelic exchange mutants of Map would provide insight on specific gene function related to their virulence and importantly improve the potential of developing an effective, live-attenuated Map vaccine.
Tuberculosis vaccination. Bacille Calmette-Guérin (BCG), developed in the 1930's, is a vaccine against tuberculosis that is prepared from an attenuated strain of live bovine tuberculosis bacillus, Mycobacterium bovis, that has lost its virulence in humans by being specially cultured in an artificial medium for years. BCG is regarded as among the safest and most widely used vaccines in the world, and remains the only vaccination available against tuberculosis. The bacilli have retained enough antigenicity to become a somewhat effective vaccine for the prevention of human tuberculosis. BCG vaccine is at best 80% effective in preventing tuberculosis for a duration of 15 years, however, its protective effect appears to vary according to geography. It is used because it is effective in reducing the likelihood and severity of TB in infants and young children, particularly in areas of the world where TB is highly prevalent, and the chances of exposing an infant or young child are high. In the United States BCG is not used, because TB is not prevalent and the chances are small that infants and young children will become exposed. Additionally, BCG may cause a tuberculin skin test to convert from negative to positive, which is confusing because the TB skin test (Mantoux test) is the best available test for TB infection, and widespread use of BCG would make the skin test less useful.
BCG is efficacious against tuberculous meningitis in the pediatric age group, but its efficacy against pulmonary tuberculosis appears to be variable. The most controversial aspect of BCG is the variable efficacy found in different clinical trials that appears to depend on geography. BCG seems to have its greatest effect in preventing miliary TB or TB meningitis, for which reason, it is still extensively used even in countries where efficacy against pulmonary tuberculosis is negligible. Other recognized uses of BCG include, but are not limited to, use in protecting against leprosy, Buruli ulcer, and in cancer immunotherapy (e.g., superficial forms of bladder cancer, immunotherapy of colorectal cancer, and for the treatment of equine sarcoid in horses), type I diabetes, and interstitial cystitis (IC)/painful bladder syndrome (PBS) (chronic inflammatory bladder problems with unknown etiology). There is, therefore, a pronounced need in the art for novel, and more efficacious compositions and methods for vaccinating against tuberculosis, and other disorders.
Crohn's disease. Crohn's disease (aka regional enteritis) is a chronic, episodic, inflammatory bowel disease (IBD) and is generally classified as an autoimmune disease. The exact cause of Crohn's disease is unknown, but genetic and environmental factors have been invoked in the pathogenesis of the disease. Crohn's disease can affect any part of the gastrointestinal tract from mouth to anus; as a result, the symptoms of Crohn's disease vary among afflicted individuals. The disease is characterized by areas of inflammation with areas of normal lining between in a symptom known as skip lesions. The main gastrointestinal symptoms are abdominal pain, diarrhea (which may be bloody, though this may not be visible to the naked eye), constipation, vomiting, weight loss or weight gain. Crohn's disease can also cause complications outside of the gastrointestinal tract such as skin rashes, arthritis, and inflammation of the eye. Crohn's disease affects between 400,000 and 600,000 people in North America. Prevalence estimates for Northern Europe have ranged from 27-48 per 100,000. Crohn's disease tends to present initially in the teens and twenties, with another peak incidence in the fifties to seventies, although the disease can occur at any age. Although the cause of Crohn's disease is not known, it is believed to be an autoimmune disease that is genetically linked. Unlike the other major types of IBD, there is no cure for Crohn's disease and remission may not be possible or prolonged if achieved. In cases where remission is possible, relapse can be prevented and symptoms controlled with medication, lifestyle changes and in some cases, surgery. Adequately controlled, Crohn's disease may not significantly restrict daily living. Treatment for Crohn's disease is only when symptoms are active and involve first treating the acute problem, then maintaining remission. Treatment options are restricted to controlling symptoms, putting and keeping the disease in remission and preventing relapse.
Interestingly, a recent report by the Canadian Broadcasting Corporation describes an apparent association between Mycobacterium avium subsp. paratuberculosis (Map) and Crohn's disease, and suggests that transmission of MAP from infected cattle to humans through milk could explain much about the occurrence of Crohn's, including its geographical distribution and rising incidence.
There is, therefore, a pronounced need in the art for novel, and more efficacious compositions and methods for treating and/or preventing Crohn's disease and other inflammatory bowel diseases.